Viral erythrocytic necrosis (YEN) is a unique viral infection of marine and anadromous teleosts. Our studies have focused on VEN in cod (Gadus morhua) and herring (Clupea harengus h .) with emphasis on characterization of the virus and its interaction with the host. Ultrastructural studies in our laboratory have shown that VEN virus of cod is structurally larger (330 nm) and more complex than VEN virus in herring (145nm); both are icosohedral in shape with a central nucleoid which may or may not be toroid. The virus from herring and Greenland cod have no structures external to the capsid whereas VEN virus from cod in North America have an amorphous peplos. Horizontal transmission of the virus in cod was accomplished although the attack rate was low; intraspecific tranmission was not accomplished. Infection with VEN virus resulted in a reduced resistance to stress, but no direct mortalities were noted. The oxygen transport function of VEN infected erythrocytes was not altered. An in vitro system for long term maintenance of cod erythrocytes was developed. Further work with this system revealed that VEN infected cells of both cod and herring lysed more rapidly than did uninfected cells. Incorporation of thymidine and amino acids, but not uridine, was significantly higher. in VEN infected cells than in uninfected cells, indicating virus replication had occurred. The effect of VEN infection on several blood parameters, including serum electrolytes, formed element composition and morphology, and cellular and serum enzymes are currently being studied.

A survey was undertaken to determine the incidence of viral erythrocytic necrosis (YEN) among hatchery reared salmonids in the state of Oregon. Thirty-five different stocks of fish were examined. Included were steelhead trout (Salmo gairdneri), coho (Oncorhynchus kisutch), chum (O. keta), chinook (O. tshawytscha), and kokanee (O. nerka) salmon. Inclusion bodies typical of VEN were found in the red blood cells of fish from 9 of the 35 stocks examined. Chum salmon were most heavily infected, but inclusion bodies were also detected in blood from coho and chinook salmon and steelhead trout. Progeny of selected populations of VEN infected adults were examined and cytoplasmic inclusions were found in erythrocytes of chum and coho juveniles.

During 1978, we isolated a previously undescrived pathogenic virus from ovarian fluids of normal appearing adult landlocked masu salmon (Oncorhynchus masou) at the Otobe Salmon Hatchery, Hokkaido, Japan.Characteristics of the newly recognized virus conform to those of the Herpesvirus group, and the agent is provisionally named Oncorhynchus masou virus (OMV).The OMV has proven to be lethal for chum salmon (O keta) by artificial immersion infection. Following immersion, 80 to 150-day-old fry began to die at 11 to 12 days later, and 35 to 60% of them succumbed in the ensuing 50 days. However, no death occurred among 240-day-old chum salmon that were similarly infected.Marked histopathologic changes were observed in liver sections. These were multiple foci of severe necrosis and syncytia formation.Further repeated experiments using other species of salmonids revealed that coho salmon (O. kisutch), kokanee salmon (O. nerka), and rainbow trout (Salmo gairdneri) were also susceptible to OMV, although some variations in susceptibility were noted.From the evidence thus far obtained, OMV is clearly a new pathogen of salmonids.

Among survivors of experimental infection of herpesvirus OMV which was considered to be a new viral pathogen of salmonids producing hepatic necrosis and significant mortality, more than 60% developed tumors. The perioral maxillary and mandibular region are the most frequent site of tumor formation. In decreasing order of frequency, tumors were also found on the caudal fin, gill-cover, eye, and kidney.Histopathologically the tumors were composed of abundantly proliferative, well differentiated epithelial cells supported by fine connective tissue stroma.Although virus particles were not observed in tumor cells, OMV isolation was successful from a tumor tissue sample that appeared necrotic on day 275 postinfection and from primary cultures of a tumor tissue from another fish sampled 296 days postinfection.From the evidence thus far obtained, OMV is considered to be a new pathogenic and oncogenic salmon virus.It is the first oncogenic agent to be isolated from fish.

A new virus of the family Reoviridae has been isolated from adult chum salmon (Oncorhynchus keta) returning to the Tokushibetsu Hatchery, Hokkaido, Japan. The virus replicates in selected fish cell lines incubated between 10 and 20°C and produces a unique cytopathic effect. Electron micrographs reveal an icosahedral particle 75 nm in diameter composed of a double capsid. The outer capsid is selectively removed by α-chymotrypsin leaving a subviral particle with enhanced infectivity. The virus is resistant to pH 3 and chloroform. It is unstable at 56°C and does not hemagglutinate human type 0 erythrocytes. Infectivity is not reduced by antiserum to infectious pancreatic necrosis virus. Replication is not inhibited by 5-fluro2'-deoxyuridine. Acridine orange stain reveals typical reovirus-like cytoplasmic inclusions. The virus is not lethal for chum salmon fry, chinook salmon (O. tshawytscha) fry, or kokanee salmon (O. nerka) fry by injection. The virus replicates in the three species tested producing focal necrotic lesions in the liver of chum and chinook salmon fry.

Persistent infections were established with infectious pancreatic necrosis virus (IPNV) in chinook salmon (CHSE-214), steelhead trout (STE-137) and in rainbow trout (RTG-2) cell lines. Viral persistence was characterized by the release of infectious virus, positive immunofluorescence, resistance to superinfection with homologous virus and susceptibility to challenges with heterologous viruses.The morphology and growth characteristics of persistently infected (PI) and uninfected cell lines was indistinguishable. PI CHSE-214 and STE-137 cell lines produce little or no interferon. Activity suggesting interferon was detected in culture fluids from PI RTG-2 cells.Temperature-sensitive virus was not detected in the culture fluids of PI CHSE-214 or STE-137 cell lines and the RTG-2 line has not been tested. Virus in the culture fluids of both PI STE-137 and RTG-2 cell lines demonstrated autointerference when inoculated onto uninfected control cells. The autointerfering component from PI STE-137 cells was removed from the culture fluid by ultracentrifugation and had a bouyant density of 1.29 g/cc in a cesium chloride gradient. Infectious virus also produced by PI STE-137 cells had a density of 1.33 g/cc. The production of defective interfering (DI) virus during viral persistence was further supported by electron microscopic examination of PI STE-137 cells. The production of DI virus by PI STE-137 cells as well as interferon in the PI RTG-2 cells were proposed as two mechanisms by which the cytocidal course of infection in these cell lines is prevented.

Negatively stained EVA and EVEX with typical rhabdovirus shapes were measured using catalase crystals as the standard. Their mean sizes were 143.2×83.5 and 144.6×83.7 nm respec tively. The CPE caused by EVA and EVEX in RTG-2 cells were very similar, but they were clearly distinguishable from that of IHNV. They induced marginal hyperchromatosis, deforma of nuclei and formation of basophilic inclusions of irregular shapes in the cytoplasm of in fected cells. Finally, infected cells became round and then collapsed in pieces. By SEM observa of CPE caused by both viruses, it was found that infected cells having long filopodia became round with some shrinking. Serologically, there is close similarity between EVA and EVEX, but both are clearly different from IHNV and probably different from VHSV type II and 23-75 strain. Infectivity trials with rainbow trout fry were carried out at 10°C, 15°C and 20°C. Both EVA EVEX showed more severe pathogenicity at higher temperatures. Symptoms and histo pathological changes of fish infected with EVA and EVEX were similar to those of IHN, and included hemorrhage of skeletal muscles, intensive necrosis and hemorrhage of hematopoietictissue of the kidneys and focal necrosis of liver, spleen and panceras. The fact that EVEX showed strong pathogenicity even at 15°C makes us concerned about the introduction of a new viral disease in rainbow trout cultured in Japan.

Microscopical findings from moribund Kuruma shrimp larvae, Penaeus japonicus, showed no evidence of any polyhedral inclusion bodies in either squash preparations of the affected mid-gut gland or in preparations stained by Vago-Amargier's method.Pathological changes were remarkable cellular necrosis and collapse of mid-gut gland. It was noted that nuclear hypertrophy and collapse always followed these cellular changes.Electron microscopical findings showed cytoplasmic collapse of mid-gut gland cells and the nuclear hypertrophy resulting in karyorrhexis, as well as virions and incomplete viral particles representing virogenic stages in the affected nuclei. The average length and diameter of virions was 310 nm and 72 nm respectively, and length of the nucleocapsids was 250 nm.Infectivity trials carried out employing oral and water borne inoculations revealed high cumulative mortality to healthy shrimp larvae.

A permanent cell line has been established from an aflatoxin-induced hepatoma of rainbow trout. The cell line is now 17 years old and has been carried through 133 serial passages. Chromosome analysis indicates the cells are heteroploid and show a bimodal distribution with modal numbers of 54 and 60. Cells are routinely cultured at 18°C and are easily stored in liquid nitrogen.They are susceptible to both the viruses of infectious hematopoietic necrosis and infectious pancreatic necrosis. It is not known if these cells have undergone malignant transformation. However, numerous attempts to cultivate normal liver cells have failed. This culture may represent the first salmonid cell line derived from tumorous tissue.

Cell lines were begun from kidney tissues of yellow tail (Seriola quinqueradiata) and sea bass (Lateolabrax japonicus), and from the fry of red sea bream (Chrysophrys major). The cells are grown in MEM-20 enriched with NaCl, incubated at 20°C and subsequent transfers maintained.Cell lines derived from the kidney of yellow tail, designated YTK, have been subcultured 64 times since its initiation on July 13, 1977, from the fry of red sea bream, designated RSBF, have been subcultured 28 times since its initiation on May 19, 1978, and from the kidney of sea bass, designated SBK, have been subcultured 31 times since its initiation on October 11, 1978.The cells were tested for growth curve and sensitivity to the freshwater fish viruses IPNV and IHNV.

Etiological study was carried out on abnormally enlarged ovary of Japanese oyster, Carssostrea gigas (THUNBERG).Light and electron microscopic examination revealed that follicle cells and ova of abnormal ovary contained one or more inclusion bodies in addition to their own nuclei. These inclusion bodies were observed to grow with the development of the ova. The body is covered with electron dense wall and contained mitochondria, endoplasmic reticulum, granuels of various size and various electron density and nuclear body with a nucleolus like body.The above mentioned structure clearly indicates that the inclusion body is a parasite possibly belonging to Subclass Coccidiida.

A description is given of a new myxozoan parasite located in the intestinal wall of the cultured carp Cyprinus carpio from Japan. The name Thelohanellus kitauei n. sp. is suggested for this parasite whose spores are characterized by an egg-shaped balloon-like sack, 33.4μm by 15.0μm in size in average. Spores measure 26.3μm in length and 9.2μm in breadth and thickness in average in water. The polysporous vegetative form which forms a giant polyp-like swelling in the intestine is inferred to be a single winding tube based on histological observations of the serial paraffin sections of the swellings. A mass of minute spherical disporous form is exceptionally observed in a small visible lesion lying under the mucosa, too.

Following the treatment of 1 ppm Dipterex the metacercaria of yellow grub Clinostomum complanatum (RUD., 1819) which parasitized the cultivated ayu in Taiwan could excyst from the muscle, penetrate into the internal organs and initiate death of the fish.Loach and ayu were demonstrated to be the second intermediate host, the Radix snail is its first intermediate host. In the in vitro culture system, the metacercaria could maintain their activity up to three months and still possess infectivity, but could not reach the mature stage.Following 2-3 days ingestion of metacercaria by herons, the final host, the adults were found in the esophagus and mouth cavity.

Saprolegniaceous fungi associated with the rainbow trout (Salmo gairdneri) were observed at Ma-ling Hatchery in Tai-Chung Prefecture, Taiwan during December, 1979 to February, 1980. One strain among the isolated fungi attached a male spawning fish was identified as Aphanomyces laevis. Another strain infected mainly on the peduncle and caudal fin of a female spawning fish was examined and identified as Saprolegnia diclina. The former was incubated at 10°C and gave well-developed sexual organs, and the latter was kept at 16°C and appeared both asexual and sexual structures. It may produce abundant of antheridia and oogonia either short after or after prolonged period of time when mycelium was removed from diseased fish. Oogonial wall of this fungus is usually pitted under point of attachment of antheridial cell and shows antheridia of diclinous form only. Some isolates of S. dicline were heavily affected by Woronina polycystis (in the Order Plasmodiophorales) which acts as an obligate parasite and eventual destruction of host hyphae.

The immersion method for the immunization of eel (Anguilla japonica) with the pathogen, Edwardsiella anguillimortifera, was studied and the results are summarized as follows:(1) Using the immersion method the immune response of eels can be induced effectively.(2) The effect of the treatment of hyperosmotic solution on the immunization of eels against E. anguillimortifera was evaluated. Following hyperosmotic treatment in 5.32% NaCl bath for 3 min, the eels were then immunized by soaking in the bacterial suspension for 3 min with heatkilled or formalin-killed Edwardsiella. The maximum titers in 5.32% NaCl pretreated eels were 1024 and 256 respectively, at fifth week after immunization. However, the maximum titers in the other two groups without osmotic shock were 256 and 128 respectively, at fourth week and seventh week after immunization.(3) The cumulative mortality of the eels against E. anguillimortifera challenge was higher in both groups with hyperosmotic pretreatment even though both had higher antibody titers.(4) The opsonic index of serum of immunized eels was 5.29.(5) In comparison with the control eels, the phagocytosis of the peritoneal leukocytes of the immunized eels increased significantly and specifically.

Both orally immunized ayu, Plecoglossus altivelis, and control fish were challenged with organism, Vibrio anguillarum, by three ways. First, fish were exposed to organism discharged from naturally infected fish for 24 hr. Organism was isolated at high percentage from almost all part of body in the control fish, while the isolation rate was low particularly on the body surface in the immunized fish.Secondary, fish were bathed in bacterial suspension of a concentration of 107 cells per milliliter for 15 min. Twenty four hours after challenge organism was isolated from the skin of the control fish, then the number of organism increased gradually for the next 72 hr. No organism was detected in the intestine or its contents in both groups neither in the skin of the immunized fish.Finally, fish were injected intramuscularly resulting almost equal mortality in both groups. Agglutinin titer in the body surface mucus rose to 1: 64 in the immunized fish, but did not occur in the control fish. Agglutinin titer rose in neither serum nor intestinal mucus in both groups. The body surface mucus of the immunized fish prevented organism from adhering to the skin more effectively than that of the control fish did.From these results it is assumed that the defence effect by the oral immunization is attributed mainly to the agglutinin secreted in the body surface mucus.

As a basic study on the development of vaccine for pseudotuberculosis in cultured yellowtail, Seriola quinqueradiata, the present paper describes our investigations into humoral aspects of immune response and efficacy of experimental vaccination against this disease in yellowtail.The experiments indicated that the epidemics of pseudotuberculosis in the adult yellowtail were prevented by post-infectious immunity. In addition, it was observed that yellowtail immunized by intraperitoneal injection with formalin-killed cells of Pasteurella piscicida which was the causative agent of this disease, produced substantially the same agglutinating antibody as in the case of post-infectious immunity. However, only macroglobulin antibodies with agglutinating antibody activity in serum were detected.Vaccination by oral, immersion, and spray methods that were practical for mass vaccination of fish were applied to pseudotuberculosis in yellowtail. Efficacy of vaccination by these methods were compared to the one by injection method, and the result shows that both are almost highly the same in efficacy. It was observed that agglutinating antibody was concentrated in the tissue stimulated with antigen.

The usefulness of the bacteriophages as a biological control agent for the culture fish diseases is based on the following reasons: (i) The growth rate of bacteriophage is much faster than bacteria; (ii) The infection of bacteriophage occurs in aqueous state; (iii) The infection of bacteriophage is very specific in respect to its host; (iv) There is no problem of drug residues and drug toxicity as with the use of chemotherapy. We have initiated the isolation of bacteriophages to infect Aeromonas hydrophila which is the pathogen of eel's red-fin disease. Among the eight isolated bacteriophages, AH1 has strongest bacteria-lysis ability. Therefore, AH1 was selected as the experimental model system for the study of biological control of diseases. The one-step growth curve showed that AH1 started to form phage particles after 50 min of infection and completed at 100 min with a burst size of 160. It means that one AH1-infected A. hydrophila can produce 160 phage particles. The A. hydrophila concentration was reduced 2×106 times compared with the starting concentration after infection with phage AH1 at an M.O.I. =1.2. In order to test the loss of pathogenecity of A. hydrophila after AH1 infection, the AHl-infected bacteria were injected to loach Misgurnus anguillicaudatus. After 3 hr infection of AH1, the A. hydrophila had completely lost its infectivity and mortality in the injected loaches. Even the multiplicity of infection (ratio of bacteriophages to bacteria) lowered to 0.001, the infectivity and mortality were reduced to 40 % of uninfected A. hydrophila. The bacteriophage AH1 viral particles were very stable in sterilized tap water, less stable in fish pond water and most unstable in distilled water.

One hundred sixty eight strains of Edwardsiella tarda collected from cultured eel (Anguilla japonica), tilapia (Tilapia nilotica) and channel catfish (Ictalurus punctatus), and from water and plankton of culture ponds were studied for their sensitivities to 10 chemotherapeutic agents: chloramphenicol (CM), tetracycline (TC), streptomycin (SM), kanamycin (KM), aminobenzyl penicillin (ABP), cefazolin (CEZ), nalidixic acid (NA), furazolidone (NF), sulfamonomethoxine (SA) and trimethoprim (TMP). All strains were highly sensitive to CEZ and TMP. Only 32 of 168 strains were sensitive to all the drugs tested. The remaining 136 strains were resistance to various combinations of the 8 drugs (CM, TC, SM, KM, ABP, NA, NF and/or SA). The most common types of drugs resistance were to combinations of CM, TC, NF and/or SA.Transferable R plasmids were detected in 38 out of 136 resistant strains. The most common type of resistance markers of R plasmid was CM, TC and SA. Other R plasmids had markers for resistance to TC, SA, TC SA and CM TC SM KM SA.Cryptic plasmids were also detected in all strains E.. tarda tested by agarose gel electrophoresis.

The widespread nature of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD) among salmonid fish, was demonstrated in trout and salmon samples taken over the past 20 years from freshwater streams and salt water sites located on Cape Breton Island, Nova Scotia, Canada. Although ubiquitous in this area, major mortalities have been reported from only a few isolated locations. However, heavy losses do occur when infected fish are acclimated to sea water prior to their salt water phase of life. The diagnosis of bacterial kidney disease, which once depended on the Gram stain of a kidney smear, now utilizes more sensitive, serological confirmation techniques such as the fluorescent antibody technique or precipitin test.In Atlantic salmon smolts, this disease exists in many internal organs but bacterial numbers concentrate in the kidney and proliferate as the temperature surpasses 4°C in the Spring. In advanced infections, lesions or bacterial pustules form on several organs but most often on the kidney.We have investigated vaccination and nutrition as possible prophylactic methods to minimize BKD infection occurrence and severity. In vaccination trials, post yearling Atlantic salmon parr administered a 0.1 ml intraperitoneal injection of formalin killed BKD cells emulsified in Freund's complete adjuvant showed an elevated agglutinating antibody response and almost complete absence of BKD lesion formation in the kidneys. In the nutritional studies, a reduction in the incidence of BKD infections with lesions was observed in the July sample among fish fed diets with a high concentration of trace elements or a low calcium content. This therapeutic effect was attributed to the increased availability of trace elements for metabolic purposes.

This paper reports on experiments undertaken to determine whether fluorescent antibody (FA)-based diagnoses of the Renibacterium salmoninarum carrier could be corroborated using a culture method. Preliminary experiments showed that culture could indeed be employed for corroborative purposes because it was more sensitive at detecting R. salmoninarum than the FA technique; the experiments indicated, however, that maximum sensitivity of the culture method was only achieved when the tissue samples were first freed of an anti-R. salmoninarum activity.Following the preliminary experiments, the corroborative value of the culture method was tested on a field scale with two lots of fish. With a stock of coho salmon (Oncorhynchus kisutch) that was suffering chronic losses due to bacterial kidney disease (BKD), culture proved to be more sensitive at detecting R. salmoninarum carriers than the FA method. With these fish, the method was clearly of corroborative value. With sockeye salmon (O. nerka) that had never had a history of BKD but which possessed anti-R. salmoninarum agglutinins and which were FA-positive for R. salmoninarum, the culture method proved to be of no corroborative value. R. salmoninarum was not isolated even when the sockeye were temperature-stressed and treated with prednisolone acetate to unmask carried pathogens. These observations raise the question as to whether fish carry more than one type of bacterium resembling R. salmoninarum. They also raise concerns about how FA-based diagnoses of the R. salmoninarum carrier should be interpreted. Further studies to investigate these points are indicated.

Streptococcal infections have been frequently observed in cultured freshwater fish, tilapia (Tilapia nilotica), rainbow trout (Salmo gairdneri) and ayu (Plecoglossus altivelis) at farms in various districts of Japan. The causative agents isolated from diseased tilapia, rainbow trout and ayu had the same morphological as well as biochemical characteristics. All the strains were also serological homogeneous. These strains were found to be pathogenic to freshwater fish after intraperitoneal injection. The autoclaved and hot-HCl treated cells did not react with any of the group specific sera used: Lancefield A, B, C, D, E, F, G, H, K, L, M, N, O and MG. This organism was not identical to any strains of Streptcoccus previously reported.

Gliding bacteria inflicted fish diseases were prevailing in November and December, but not in May and June. Annual survey of the gliding bacteria revealed that Flexibacter columnaris was common in September and October while other Flexibacter spp. in March and April.The virulence of these bacteria to eel and tilapia were examined. Methods and conditions for their infection were also studied. In running water, F. columnaris was more virulent to eel than to tilapia, while Flexibacter spp. were more virulent to tilapia than to eel. Artificial infection of gliding bacteria was more effective by contact method than by injection method. Mortality rate of fishes infected with gliding bacteria was high when they were kept in standing water rather than running water post-infection.Supplement of ferric ion to the artificially infected fishes shortened their survival time. This was more pronounced in fishes infected by injection. However, human transferrin afforded protection to fish against infection with these bacteria.

As red spot disease of eels (Pseudomonas anguilliseptica infection) has occurred restrictedly in Japan so far, a brief histroy of investigations of the disease was given before entering the main issue.In order to find practicable methods for artificial infection in Japanese eels (Anguilla japomica), injection, oral administration, addition, and immersion methods were tested in this study. As the results, the three methods except the oral administration were demonstrated to cause a mortal infection in the experimental animal. In the addition method, eels were required to be kept in salt containing water in order to be successfully infected. In the immersion method, a pre-immersion in 7 % saline for 10 min or 10 % saline for 5 min was necessary to bring a definite result.The addition technique was thought to be most applicable for challenge test in vaccinated and emaciated eels.

A total 291 strains of motile members of the genus Aeromonas were classified according to the taxonomic scheme proposed by POPOFF and VERON (1976). Forty three were indetified as A. hydrophila biovar hydrophila, 9 as A. hydrophila biovar anaerogenes, and 52 as A. sobria. The remaining 187 strains could not be identified. Thirty seven (86%) of 43 A. hydrophila biovar hydrophila, 3 (33 %) of 9 A. hydrophila biovar anaerogenes, 32 (62 %) of 52 A. sobria, and 65 (35%) of the 187 unidentified originated from fishes.All 6 A. hydrophila biovar hydrophila examined fell in the category of high virulence. The Japanese loach injected with them were killed earlier than those injected with the other motile Aeromonas, and the difference of mean time to death was statistically significant (P<0.01). The difference of average value of proteolytic activity of the broth culture filtrate between A. hydrophila biovar hydrophila and the other was significant (P<0.01), also. Exophthalumus and scale protrusion were evident only among fish injected with the sonicated cell extracts of A. hydrophila biovar hydrophila.